This project studies peripheral blood hematopoietic progenitors (PBHP) as a target for gene therapy of inherited diseases affecting the function of human phagocytic cells, including neutrophils, eosinophils, and monocytes. A related goal of this project is development of novel cellular therapies based on genetically engineering human PBHP and their phagocytic cell progeny to endow them with new properties to augment host defense against chronic infections with intracellular pathogens including tuberculosis and other mycobacteria infections. We have defined conditions for optimum harvest, purification and culture of the primitive human hematopoietic cells with a CD34 surface antigen phenotype. In a clinical study we determined the optimum dosing of subcutaneous granulocyte colony stimulating factor (G-CSF) to recruit primitive CD34 progenitor cells from bone marrow to peripheral blood. We showed that mobilization is impaired in patients with chronic granulomatous disease and ADA-deficient SCID. We have studied optimum conditions for growth of CD34+ cells in culture, developing conditions leading to predominantly neutrophils, eosinophils or monocyte/macrophages. This will be important for studies aimed at engineering new characteristics into specific end stage phagocytes by gene transfer into progenitors. Using conditions for monocyte differentiation we introduced a gene for interferon gamma (IFN-gamma) into these cells by targeting the CD4+ progenitors, finding that this resulted in augmentation of oxidase activity and an increase in IgG Fc receptors. In other studies, the gene for nitric oxide synthase (NOS) was transferred into the human erythroleukemia cells line, finding that the constituitive nitric oxide production resulted in changes in post-translational regulation of several genes related to iron metabolism and hemoglobin production. This may provide insight into the mechanisms responsible for anemia of chronic infection. In studies of transplantation in mice, we have demonstrated that a combination of growth factor administration followed by low dose radiation is an effective method for enhancing engraftment of transfused congenic progenitor cells. This has important implications for engraftment following gene therapy in humans targeting autologous CD34+ cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000644-05
Application #
2566839
Study Section
Special Emphasis Panel (LHD)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
1996
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
De Ravin, Suk See; Li, Linhong; Wu, Xiaolin et al. (2017) CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease. Sci Transl Med 9:
Sweeney, Colin L; Teng, Ruifeng; Wang, Hongmei et al. (2016) Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis. Stem Cells 34:1513-26
De Ravin, Suk See; Reik, Andreas; Liu, Pei-Qi et al. (2016) Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol 34:424-9
Merling, Randall K; Sweeney, Colin L; Chu, Jessica et al. (2015) An AAVS1-targeted minigene platform for correction of iPSCs from all five types of chronic granulomatous disease. Mol Ther 23:147-57
Zou, Jizhong; Sweeney, Colin L; Chou, Bin-Kuan et al. (2011) Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting. Blood 117:5561-72
Kang, Elizabeth M; Malech, Harry L (2009) Advances in treatment for chronic granulomatous disease. Immunol Res 43:77-84
Sweeney, Colin L; Malech, Harry L (2009) Functional neutrophils from human ES cells. Blood 113:6503-5
De Ravin, Suk See; Malech, Harry L (2009) Partially corrected X-linked severe combined immunodeficiency: long-term problems and treatment options. Immunol Res 43:223-42
Chinen, Javier; Davis, Joie; De Ravin, Suk See et al. (2007) Gene therapy improves immune function in preadolescents with X-linked severe combined immunodeficiency. Blood 110:67-73
Shearer, William T; Malech, Harry L; Puck, Jennifer M (2007) Primary immunodeficiency: meeting the challenges. J Allergy Clin Immunol 120:753-5

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